The papillomaviruses induce benign, proliferative squamous epithelial lesions in their natural hosts and an only undergo vegetative replication in terminally differentiated epithelial cells. This requirement has hampered the study of the complete viral life cycle because of the difficulties in generating a differentiated stratified epithelium in tissue culture. To date, most analyses of papillomavirus gene functions have been carried out in cell lines which are probably analogous to the basal cells of a papillomavirus-infected lesion. Such studies have shown that multiple proteins encoded by the bovine papillomavirus E1 and E2 genes are required for regulation of viral DNA replication and transcription. We have taken two approaches to learn exactly how the virus uses the various E1 and E2 proteins to regulate the complete viral life cycle in infected skin lesions: (1) Using a combination of organotypic raft cultures and xenografts on nude mice we have developed systems in which we can generate fully differentiated bovine and human epithelium. We have also developed gene transfer techniques in order to introduce mutated papillomavirus genomes into epithelial lesions with high efficiency. (2) Using antibodies specific for the various E1 and E2 proteins, we are examining in which epithelial layers of naturally-occurring lesions the different gene products are expressed.